Non Technical Summary Crop pests are estimated to cause the loss of up to 40% of the world's crops annually, at an estimated cost of US $200 billion. At the same time it is predicted that pesticide sales will rise in the US to $33 billion by the year 2003, about 75% of which will go for agricultural applications. Chemical pesticides are both expensive and also raise concerns regarding contamination of food and environment. The development of alternative methods of plant pest control is crucial to sustainable agriculture, particularly in the desert southwest, where drought conditions can lead to total devastation of crops under attack by plant pests. The purpose of this research is to develop alternative methods for pest control by applying the biotechnologies of genetic engineering, gemomics, and proteomics. These advancing technologies can be complemented with related basic and applied sciences, improvements in overall pest management strategies, and the continuation of appropriate
traditional agricultural practices to provide better approaches for plant pest control. The primary purpose of this project is sustainability of agriculture, particularly in arid and semi-arid regions, through the use of advanced sciences to both protect plants from pest invasion and to protect the earth's ecosystems from further damage by chemical pesticides.

Goals / Objectives The objective of this research is to use molecular biology techniques (genetic engineering) to develop specific pesticidal capabilities in plants of agronomic importance. Genetic engineering and related sciences offer an alternative approach for the control of crop damaging pests. The development of alternative pesticides through biotechnology will help to reduce the current reliance on expensive and environment comprising synthetic chemicals to control plant pests. Genetic engineering follows the methodology of classical breeding for desirable traits, but provides a means to broaden the available gene pool, increase the number of potential recipient plants, precisely target a plant pest and eventually shorten the time frame required to achieve the desired goal of economical protection of plants from devastation by plant pests. This devastation is more seriously felt in times of drought, particularly in the desert lands such as the southwest US, when plants are less
capable of recovery from pest attack.

Project Methods The approach taken for this research involves the identification of natural pesticides, such as Bacillus thuringiensis toxins and proteases, the isolation and identification of the genes which express these pesticides, and the engineering of these genes with various sequences for signal peptides and promoters to enhance the efficacy of the pesticidal gene in transgenic plants. The use of proteomic techniques will be added in an attempt to identify new pesticidal genes and the genes for proteins which may be involved in enhanced expression. The specific approaches of this research include: 1. the identification of proteins with natural pesticidal activity from biological sources including plants, animals, bacteria, and fungi, 2. engineering of genetic constructs designed to improve gene expression and effectiveness in transgenic plants, 3. transformation of target agronomic crop plants, 4. field trials to test efficacy, and 5. commercial transfer of engineered
pesticidal genes for application in integrated pest management.

Progress 09/01/03 to 08/31/05

OutputsDuring the past year significant progress has been made on several fronts. Significant new developments include: 1) the initiation of work on novel Bt toxin derivatives that may have activity against nematodes; 2) demonstration that a novel class of resistance genes derived through combinatorial chemistry methods are active against pest transmitted diseased in transgenic plants; 3)development of a novel system for analysis of cap-independent translation enhancers; 4) the cloning of a colagenase gene from C. elegans for use in engineering nematode resistant plants; and 5) the application of molecular phylogeny techniques in high resolution pest identification in cases where traditional methods are unable to quickly and easily distinguish between different agricultural pests. We have also continued work on ongoing projectcts including our long term work aimed at developing and optimizing technology for the simultaneous delivery of multiple pesticide genes to transgenic
plants.

ImpactsCrop pests are estimated to cause the loss of up to 40% of the world's crops annually, at an estimated cost of US $200 billion. Advances from this research will reduce the impact of pests and the diseases they spread, thereby increasing agricultural productivity. In addition, the control of pests and the diseases they transmit will improve the sustainability and profitability of agriculture will also be improved by reducing the reliance on chemical pest controls. Our current work on molecular phylogeny resulted in two papers published and provided a direct benefit to local producers by allowing definitive identification of pest problems, including one which distinguishes between local and exotic fire and species which is extremely important to local growers since the two types are difficult to tell apart morphologically and since the presence of exotics results in a quarantine on agricultural products at substantial cost to local producers. Our continuing work on
nematode resistance genes, including the cloning of C. elegans colagenase and new work on novel Bt toxins, will be crucial since robust nematode control options are not emerging while past options like MeBr are being removed. Environmentally sound control of insect pests through biotechnology will have a tremendous impact long term. In the shorter term, some of the virus resistance genes, particularly those for geminiviruses, are expected to have a profound impact on crop production, particularly as there are currently no effective control measures against this group of viruses in many cropping systems.

Publications

No publications reported this period

Progress 01/01/04 to 12/31/04

OutputsDuring the past year significant progress has been made on several fronts. We have continued to develop multi-gene expression vectors to express multiple pesticide genes by expressing them as poly-proteins lined by self-hydrolyzing P2A peptide sequences, including construction of cassettes for the expression of multiple Bt proteins for targeting of multiple pests. This research is continuing with the synthesis and incorporation of nematode specific Bt genes into transgenic plants. The effectiveness of Bt toxins for pest control is directly related to their concentration in engineered plants. We have been pursuing long term projects aimed at understanding the mechanisms of seed storage protein body formation with the goal of understanding how these proteins accumulate to such high levels in order to increase Bt toxin accumulation in transgenic plants. Recent progress includes publication of one paper and submission of a second on mechanisms of zein protein body
formation. Control of pest transmitted diseases is also an important goal of this project. Work during the prior year focused on development of engineered resistance strategies for Beet Curly Top virus, an insect (leaf hopper) transmitted virus which is one of the most serious disease problems in pepper and tomato production in the Southwester US. We have used combinatorial chemistry techniques to create several non-virus derived resistance genes that express small RNA molecules which interfere with geminivirus replication. One highly attractive feature of these molecules is that they target a highly conserved region within the geminivirus origin of replication and are therefore likely to be broad spectrum geminivirus replication inhibitors. The generation of broad spectrum geminivirus resistance genes would be a very important achievement since this is a large and diverse family of viruses that would be impractical to control if it were necessary to make a specific resistance gene
for each individual geminivirus. We expect to publish these results in the coming year. Continuing research on virus resistance genes includes the construction of several gene silencing and dominant negative replicase resistance genes that are currently being analyzed. Some difficulty has been experienced in this work. In particular, control experiments with gene silencing constructs used to silence the GUS reporter gene (used to validate our constructs) have been either highly variable or only moderately effective. This inconsistency is an emerging trend in gene silencing and the reasons for it are not entirely clear. We have spent considerable time during the past year optimizing vector and silencing cassette design to improve the consistency and efficiency of our gene silencing systems to improve their function in virus resistance engineering work. In a related project, development of an efficient gene silencing system will allow the analysis of host genes involved in pest
interactions, particularly those involved in supporting colonization of host plants by nematodes.

ImpactsEnvironmentally sound control of insect pests through biotechnology will have a tremendous impact long term. In the shorter term, some of the virus resistance genes, particularly those for geminiviruses, are expected to have a profound impact on crop production, particularly since there are currently no effective control measures against this group of viruses in many cropping systems. Additionally, the application of new advances in molecular techniques is leading to the development of improved pest and pathogen detection systems by our group which will improve agricultural production indirectly by improving pest and pathogen identification and forecasting.

OutputsDuring the past year significant progress has been made on several fronts. We have continued to develop multi-gene expression vectors to express multiple pesticide genes by expressing them as poly-proteins lined by self-hydrolyzing P2A peptide sequences. We also began characterizing viral cap independent translation enhancers for use in making polycistronic expression cassettes by creating and validating a reporter gene cassette for the analysis of cap independent translation activity. Progress was also made in developing gene silencing cassettes for the control of pest spread diseases, with beet curly top virus being the first target. A transient infection and resistance gene analysis assay was developed and will be used for the analysis of resistance genes.

ImpactsCrop pests are estimated to cause the loss of up to 40% of the world's crops annually, at an estimated cost of US $200 billion. Advances from this research will reduce the impact of pests and the diseases they spread, thereby increasing agricultural productivity. In addition, the control of pests and the diseases they transmit will improve the sustainability and profitability of agriculture will also be improved by reducing the reliance on chemical pest controls.